The long term objective of this project is to elucidate the mechanisms of intracellular transport of proteins in eukaryotic cells. Biochemical and genetic evidence demonstrates that small, ras-like, GTP-binding proteins are involved in this process. The objective of this proposal is to identify and functionally characterize (a) GTP-binding protein(s) involved in the transport of proteins through the Golgi. The major experimental system will be a cell-free transport assay that measures the transfer of proteins between cis and medial Golgi compartments. The non-hydrolysable analog GTPgammaS inhibits transport in the cell-free system; current evidence suggests that this effect is mediated by the binding of a cytosolic GTP-binding protein to an effector on the membrane. The specific aims of the project are to (1) Purify the GTP-binding protein using its inhibitory effect on the cell-free transport assay, (3) Use the pure protein to obtain a cDNA and specific antibodies in order to study the function of the GTP-binding protein in the transport reaction. (5) Study the regulation of nucleotide exchange on the GTP-binding protein that is responsible for its activation. (6) Identify the effector to which GGBF binds tightly on the membranes in order to study its role in transport. The cell-free transport assay provides a system with which to functionally characterize specific GTP-binding proteins. The work described in the proposal paves the way for one of the first detailed description of the mechanism of action of a small GTP-binding protein of the ras super-family.